PROJECT SUMMARY Chronic itch is a symptom of many many diseases and causes stress, loss of sleep, and a decline in quality of life. In addition to the psychological damage, constant scratching physically exacerbates skin disease by enhancing skin inflammation and promoting infection. However, the only specific treatment options are antihistamines, which mostly are ineffective. Recently, the cytokine IL-31 was identified as a new target for itch. A newly-completed Phase II clinical trial of an antibody targeting the IL-31 receptor (IL31RA) showed remarkable efficacy in reducing itch in atopic dermatitis patients. IL-31 has been proposed to act by stimulating a subset of neurons in the skin that specifically transmits itch signals, and which is characterized in mice by expression of a protein called MAS-related G protein- coupled receptor A3 (Mrgpra3). However, while we have confirmed that Il31ra-positive neurons do exist, they do not overlap with Mrgpra3-positive neurons. However, Mrgprs may be involved indirectly, as we have preliminary evidence that IL-31 induces keratinocytes to release Mrgpr-activating substances. Thus, we hypothesize that IL-31 induces itch directly by acting on a new, uncharacterized subset of neurons, and indirectly by inducing keratinocytes to produce Mrgpr- activating pruritogens. We propose three sets of experiments to test our hypothesis. First, we will evaluate whether IL-31 induces changes in excitability and/or transcriptional programming in IL31RA-positive neurons. This could lead to long-term, persistent increases in itch sensation that may even outlast exposure to IL-31. Second, we will determine whether the substances released by keratinocytes that activate Mrgprs are members of a newly-identified class of Mrgpr ligands. We also will examine IL-31-induced itch in Mrgpr knockout mice. Third, we will determine the relative contributions of IL31RA-positive neurons and keratinocytes in IL-31-induced itch by knocking out IL31RA specifically in neurons or in epithelial cells. These will inform future research directions and may help to select drug delivery options. The data generated from our proposed experiments will have direct applications in the clinic, as the first specific anti-pruritic treatment option since antihistamines is an anti-IL31RA antibody. An understanding of how IL-31 works is essential to interpret future clinical results, to predict who will and will not benefit from anti-IL31RA therapy, to develop even more specific treatment options, and to identify other diseases that might be treated by targeting IL-31.